Connexins are now known to have dual functions, serving both as intercellular (cell-cell) channels that mediate direct signaling between neighboring cells, and as membrane channels (hemichannels) that mediate signaling across the plasma membrane. The functional significance of connexins is now widely recognized from studies of targeted gene disruptions and a number of human hereditary diseases shown linked to mutations in connexin genes. Tissue- and cell-specific patterns of connexin expression are linked to functional properties that principally include regulation of channel opening, i.e. gating, conductance and permeability. In this proposal, we use combined molecular and biophysical approaches to identify the pore- lining and regulatory elements of connexin channels and hemichannels using Cx46 and Cx50 isoforms. Cystein-substitution accessibility will be used at the level of single hemichannels to map pore-lining residues, which should provide a fundamental basis for understanding what biological signals are selected for transmission. Parallel studies will address correspondence between hemichannels and cell-cell channels. Both voltage and extracellular Ca2+ play important roles in regulating hemichannel opening and we examine underlying mechanisms of action. We also present a novel form of regulation, mediated by extracellular monovalent cations, that strongly modulates hemichannel opening. This form of regulation is particularly robust for the Cx50 isoform and is a physiologically viable mechanism for regulated hemichannel opening. We will examine the underlying mechanism of action and use the sequence similarities of Cx46 and Cx50 to localize molecular determinants. Finally, mutations in Cx46 have been shown to cause congenital cataracts in humans. A number of mutations have been recently reported that map to areas important in gating and permeability and we will undertake functional analyses to examine possible mechanisms of disease associated with Cx46 dysfunction. [unreadable] [unreadable]